Key to the activities of the tumor suppressor gene p53 is its availability/stability. Regulation of p53 stability is mediated by Mdm2 and by JNK, which target different pools of p53 for ubiquitination. JNK targeting of its associated proteins for ubiquitination occurs under non stress conditions, as shown for c-Jun, ATF2 and p53. In former studies we have demonstrated that JNK associates with p53 and that this binding contributes to p53 activity by phosphorylation of p53 on Thr81. JNK association with p53 also affects its stability in an Mdm2 independent manner, and takes place when JNK is not active as a kinase - under non-stress, normal growth conditions. The present proposal aims at addressing the mechanisms that underlie JNK's ability to target the ubiquitination and degradation of p53. Our preliminary results suggest that E6AP, which is the ligase for E6, serves as the E3 ligase for this process. On the basis of these observations, our working hypothesis is that JNK serves as an adaptor for E6AP's ability to affect p53 ubiquitination and stability. The proposed experiments will (1) Establish an in vitro system to demonstrate the role of E6AP in targeting p53 ubiquitination via JNK. (2) Determine the role of JNK/E6AP in altering the stability of p53 using cells that lack JNK or E6AP (KO or siRNA); (3) Determine the role of JNK in the context of E6-mediated p53 ubiquitination. The possible existence of tertiary complexes will be examined in vitro and in vivo. The interplay between these proteins and their implications for the JNK/E6AP association and activities will be determined; we will crystallize JNK-E6AP-p53 complex. (4) Assess the possible role of E6AP as the JNK ligase for the targeting of c-Jun and ATF2 ubiquitination and stability / activity. We will further determine the role of JNK and E6AP in recruitment of Pin1 to p53 and to c-Jun (both proteins were shown to be affected by Pin1 in a JNKphosphorylation- dependent manner).Overall, the proposed studies will characterize the long-sought cellular homologue of E6 and add new important information to our understanding of the regulation of p53 stability, while demonstrating the physiological significance of JNK targeting p53 ubiquitination.